Báo cáo y học: "Cytoplasmic dynein could be key to understanding neurodegeneration" pot

Genome
BBiioollooggyy
2008,
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214
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CCyyttooppllaassmmiicc ddyynneeiinn ccoouulldd bbee kkeeyy ttoo uunnddeerrssttaannddiinngg nneeuurrooddeeggeenneerraattiioonn
Gareth T Banks and Elizabeth MC Fisher
Address: Department of Neurodegenerative Disease, Institute of Neurology, Queen Square, London WC1N 3BG, UK.
Correspondence: Gareth T Banks. Email: ; Elizabeth M Fisher Email.
AAbbssttrraacctt
A new mouse mutation,
Sprawling
, highlights an essential role for the dynein heavy chain in
sensory neuron function, but it lacks the ability of other known heavy-chain mutations to
ameliorate neurodegeneration due to defective superoxide dismutase.
Published: 28 March 2008
Genome
BBiioollooggyy
2008,
99::
214 (doi:10.1186/gb-2008-9-3-214)
The electronic version of this article is the complete one and can be
found online at />© 2008 BioMed Central Ltd
Eukaryotic cells transport molecules, complexes and organelles
around the cell by means of energy-dependent motor
proteins. The main motor responsible for movement of
cargos to the minus end of microtubules is cytoplasmic
dynein. This is a huge multisubunit protein complex that
interacts with many intracellular pathways and whose
multifarious roles in the cell are far from being completely

understood. In neurons, dynein is the major retrograde
motor, moving cargoes from the synapse along the axon and
back to the cell body. Previous mutations in the core of this
motor - the dynein heavy chain - are known to ameliorate
neurodegeneration in mouse models of amyotrophic lateral
sclerosis (ALS). A recent paper by Chen, Popko and
colleagues [1] reporting a new mouse mutant for the dynein
heavy chain extends our knowledge of the effects of dynein
mutations on the nervous system, but the mystery of
dynein’s relation to neurodegenerative disease thickens.
Cytoplasmic dynein is a large complex of proteins whose
constituent members are the heavy chain (encoded by a
single gene), the intermediate chains (two genes), the light-
intermediate chains (two genes), and the light chains (three
genes) [2]. The precise stoichiometry of the intact complex is
not known, but at its core lies a homodimer of heavy chains.
This dimer binds to microtubules and enables dynein to
move in an ATP-dependent manner [3]. The other dynein
subunits are thought to maintain the stability of the
complex, to modulate its activity and to interact with
accessory and cargo proteins (Figure 1a) [4-10]. Cytoplasmic
dynein may also perform tasks other than transporting
cargos; for example, endosomes depend on dynein not just
for their motility, but also for their maturation, morphology
and receptor sorting [11].
The cytoplasmic dynein heavy-chain protein has a mass of
532 kDa and is encoded by a 78-exon gene, DYNC1H1; no
splice isoforms are known (Figure 1b). A Dync1h1 mouse
knockout results in no detectable phenotype in hetero-
zygotes and early embryonic lethality in null animals [12].

Two mouse mutants - Legs at odd angles (Loa) and
Cramping 1 (Cra1) - have been described previously, both of
which are due to point mutations in Dync1h1 (Figure 1b)
[13]. These single amino-acid substitutions result in similar
phenotypes: heterozygous animals show clenching of the
hindlimbs when held by the tail (Figure 1c) and an obvious
gait disorder, and homozygotes die at or before birth.
Histological studies of the spinal cord of heterozygotes
reveal a progressive loss of motor neurons. Retrograde
axonal transport as measured by the movements of a
fluorescent tetanus toxin fragment is normal in
heterozygous Loa embryonic motor neurons but is slowed
down in homozygotes [13,14].
SSpprraawwlliinngg
,, aa nneeww mmoouussee ddyynneeiinn hheeaavvyy cchhaaiinn mmuuttaattiioonn
The new mutation described by Chen et al. [1] is a radiation-
induced dominant mutation that arises from a 9-bp deletion
in Dync1h1 that changes the four residues from position
1,040-1,043 into a single alanine, and it lies close to the Cra1
mutation (see Figure 1b). Called Sprawling (Swl), the
phenotype of Swl heterozygotes (Swl/+) is strikingly
similar to the limb clenching of Loa and Cra1 hetero-
zygotes. Swl/+ mice also develop gait abnormalities and
have reduced hindlimb grip strength. But although the
outward phenotype of Swl heterozygotes is so similar to
those of Loa and Cra1 heterozygotes, Chen and colleagues
[1] found no reduction in the number of motor neurons in
the spinal cord of Swl/+ mice (Table 1). Instead they
uncovered clear signs of moderate sensory neuropathy.
Thus, this paper highlights for the first time the essential

role of the dynein heavy chain in the functioning of
mammalian sensory neurons.
On further examination, the authors also found a similar
sensory deficit in Loa/+ mice, and went on to show that
while nociception (the sensing of pain) was unaffected,
proprioception (the reception of stimuli produced within the
body) was markedly affected in both Swl/+ and Loa/+
strains, with a striking decrease in the number of proprio-
ceptive sensory receptors. They also found that neuron loss
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FFiigguurree 11
Heavy-chain dynein mutations.
((aa))
A schematic diagram of the cytoplasmic dynein complex. The core of the complex comprises a homodimer of heavy-
chain subunits (DYNC1H1), the carboxy-terminal half of which form seven AAA-ATPase domains (labelled 1 to 6 and C). The dynein intermediate
(DYNC1I) and light-intermediate (DYNC1LI) chains bind to the amino-terminal domain of the heavy chains. The light chains (DYNLRB, DYNLT and
DYNLL) all bind to the intermediate chains. The dynactin complex (not shown) binds to the cytoplasmic dynein intermediate chains. Adapted from [2].
((bb))
Protein domain map of the cytoplasmic dynein heavy chain, showing the location of the mutations
Loa
,
Cra1
and

Swl
. The motor domain consists of
the six known AAA-ATPase domains (AAA 1 to 6) and an unrelated seventh domain (AAAC). The microtubule-binding domain lies between AAA4 and
AAA5. The amino-terminal half of the protein contains the intermediate (DYNC1I), light-intermediate (DYNC1LI) and heavy (DYNC1H1) chain binding
domains [21,22]. The
Loa
mutation falls within both the DYNC1H1 dimerization and DYNC1I binding domains. The
Cra1
and
Swl
mutations fall outside
of the DYNC1I binding domain, but still within the DYNC1H1 dimerization domain.
((cc))
The hind-limb clasping phenotype of
Loa
/+ mice. When held by
the tail, wild-type (+/+) mice splay their hind legs away from their body. In contrast,
Loa
/+ mice withdraw their hind limbs, pulling them into their body.
Swl
/+ mice display a similar phenotype.
AAA1
DYNC1H1 dimerization
AAA2 AAA3 AAA4
Motor domain
Stalk
(MT binding)
AAA5 AAA6 AAAC
Stem domain
1866

0
2097
300
1137
2178
2450
2554
2803
2897
3187
3166
3498
3551
3780
4003
4219
4400
4644
+/+ Loa/+
Microtubule
6
C
1
2
3
4
5
MT
6
C

1
2
3
4
5
MT
DYNC1H1
DYNC1I
DYNC1LI
DYNLRB
DYNLT
DYNLL
(a)
(b)
(c)
Wild type: 576 ANEMFRIFS 584
Loa : 576 ANEMYRIFS 584
Wild type: 1051VWLQ
Y
QCLW1059
Cra1: 1051 VWLQ
C
QCLW1059
Wild type: 1036 SAVMGIVTEVEQ 1047
Swl : 1036 SAVMA EVEQ 1047
DYNC1I binding
DYNC1LI binding
in the dorsal root ganglia was greater in lumbar spinal cord
than in the cervical region and that this loss was considerably
greater for proprioceptive than for nociceptive sensory

neurons. Furthermore, there was degeneration of muscle
spindles during late embryonic development that was
concomitant with the loss of lumbar proprioceptive neurons
in Loa/+ and Swl/+ mice, and the dorsal roots of the lumbar
segments were also thinner than the ventral roots. Chen et
al. [1] conclude that the early-onset proprioceptive sensory
defect is common to Swl/+ and Loa/+, and that this defect,
rather than the motor neuron loss, is likely to account for the
movement disorder observed in both mice.
TThhee ddyynneeiinn hheeaavvyy cchhaaiinn aanndd hhuummaann aammyyoottrroopphhiicc llaatteerraall
sscclleerroossiiss
The new Swl mutation may also help us to a better
understanding of the possible involvement of dynein in
neurodegenerative disease. The devastating human neuro-
degenerative disorder amyotrophic lateral sclerosis (ALS)
involves progressive loss of motor neurons, resulting in
complete paralysis and death, usually 3-5 years after
diagnosis. The disease strikes people in mid-life and is
inexorable and incurable. Mental faculties are usually spared
while the body becomes progressively immobilized. ALS
clearly has a genetic component, but as yet only one major-
effect gene is known, superoxide dismutase 1 (SOD1), which
encodes an enzyme that removes free radicals (reviewed in
[15]). ALS-associated mutations in SOD1 are almost all
autosomal dominant with high penetrance; the enzymatic
activity of the protein generally remains intact and the
mutant protein takes on a dominant gain-of-function, which
for unknown reasons kills motor neurons.
In working with the mouse as a model system, we have the
ability to set up crosses and see what happens. Chen et al. [1]

made crosses between their Swl heterozygotes and a
SOD1
G93A
transgenic strain that models human ALS [16],
and between Loa heterozytoes and the SOD1
G93A
strain.
They report that the survival time of the Loa, SOD1
G93A
double heterozygotes is increased, as found in our previous
work on this cross [14], but that the Swl, SOD1
G93A
double
heterozygotes had no difference in survival time compared
to their SOD1
G93A
littermates [1]. The difference between the
effects of the Loa and the Swl mutations when combined
with the SOD1
G93A
transgene is intriguing, and, as Loa also
/>Genome
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TTaabbllee 11

CCoommppaarriissoonn ooff
LLooaa
//++,,
CCrraa11
//++ aanndd
SSwwll
//++ mmiiccee
Loa
/+ [1,13,20]
Cra1
/+ [13]
Swl
/+ [1]
Mutation F580Y Y1055C [GIVT]1040[A]
Lifespan Normal Normal Normal
Progressive phenotype Mildly Mildly No
Limb clenching Yes Yes Yes
Gait abnormalities Yes Yes Yes
Forelimb grip strength Reduced Unknown Normal
Hindlimb grip strength Reduced Reduced Reduced
Muscle pathology Normal Abnormal Normal
Loss of muscle spindles Yes Unknown Yes
Nociception * Unknown No
Proprioception Abnormal Unknown Abnormal
H reflex Absent Unknown Absent
Loss of lumbar DRG neurons Yes Unknown Yes
Loss of cervical DRG neurons Mild
†
Unknown No
Size of ventral root Normal Unknown Normal

Size of dorsal root Thin Unknown Thin
Diameter of sciatic nerve Thin
‡
Unknown Thin
Loss of alpha motor neurons in spinal cord Mild Mild No
Attenuates
SOD1
G93A
Yes Yes No
*Tendency to longer time in tail-flick test, but never shown to be statistically significant (Rogers D, EMCF, Martin JE, unpublished data).
†
Not statistically
significant.
‡
(Bros V, EMCF and Greensmith L, unpublished data).
causes loss of motor neurons as well as a sensory neuron
defect, one interpretation of these findings is that the
different dynein heavy-chain mutations are differentially
affecting pathways in different types of neurons.
The ability of the Loa and Cra1 mutations to attenuate the
SOD1
G93A
phenotype and extend lifespan [14,16] is still much
of a mystery. In the case of Loa, the double heterozygotes
lived for around 28% longer than their SOD1
G93A
parents
and siblings, and, bizarrely, the rate and flux of retrograde
axonal transport were actually increased compared with
their siblings. Research investigating interactions between

cytoplasmic dynein and mutant SOD1 includes reports of co-
localization of dynein components and mutant SOD1 in ALS
mouse models [17], the interaction of mutant SOD1 proteins
with cytoplasmic dynein [18] and perturbation of transport
of mitochondria in motor neurons from SOD1
G93A
mice [19].
Given that Swl has no detected motor neuron involvement
and does not attenuate the effects of the mutant SOD1
protein, one exciting possibility arising from the new work
[1] is that further insight into the different effects of the
various dynein heavy-chain mutations may well help our
understanding of SOD1-related ALS in humans (Table 1).
There is at present no obvious explanation from the sites of
the Loa, Cra1 and Swl mutations in the dynein gene to why
two out of three of them affect the SOD1
G93A
phenotype, and
the differences between these mice and the molecular mecha-
nisms of each mutation clearly warrant closer examination.
One intriguing question is whether effects on axonal
transport in motor neurons is responsible for this
differential effect on the SOD1 mutant phenotype, and a
dissection of axonal transport in live Loa/+ mice would be of
great interest in this context. Chen and colleagues [1] suggest
that altered Trk signaling may lead to cell death in Loa/+
and Swl/+ mice, raising the question of how cell signaling
pathways are altered in these mice in sensory and motor
neurons. A further question is whether the Swl, Loa and
Cra1 phenotypes arise from dysfunction of the complete

cytoplasmic dynein complex, or from an as yet unknown
function of only the heavy chain. It is likely that this huge
protein has more functions that we yet know of. Finally,
Chen et al. [1] have clearly shown that the ubiquitously
expressed cytoplasmic dynein heavy chain is essential for the
development and function of a subset of neurons in the
sensory nervous system. Why this should be remains a
mystery. For all those interested in dyneins, axonal retrograde
transport, the nervous system and neurodegeneration, there
is an exciting road ahead.
AAcckknnoowwlleeddggeemmeennttss
We thank the Wellcome Trust for support. We are most grateful to
Giampietro Schiavo, Brian Popko, Linda Greensmith and Majid Hafez-
parast for critical comments and helpful insights on the manuscript and
Ray Young for graphics.
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